Abstract
Inarguably, the green fluorescent protein (GFP) family is an exemplary model for protein engineering, accessing a range of unparalleled functions and utility in biology. The first variant to recognize and provide an optical output of chloride in living cells was serendipitously uncovered more than 25 years ago. Since then, researchers have actively expanded the potential of GFP indicators for chloride through site-directed and combinatorial site-saturation mutagenesis, along with chimeragenesis. However, to date, the power of directed evolution has yet to be unleashed. As a proof-of-concept, here, we use random mutagenesis paired with anion walking to engineer a chloride-insensitive fluorescent protein named OFPxm into a functional indicator named ChlorOFF. The sampled mutational landscape unveils an evolutionary convergent solution at one position in the anion binding pocket and nine other mutations across eight positions, of which only one has been previously linked to chloride sensing potential in the GFP family.